Introduction to ARV Drug Resistance

New Clinicians’ Workshop

Susa Coffey, MD Division of HIV, ID and Global Medicine

Disclosures

I have no disclosures

ARS QuestionWhich resistance test do you order for ART-naïve patients?

1. Standard genotype (RT and PR)2. Standard phenotype (RT and PR)3. Genotype + phenotype (RT and PR)4. Integrase phenotype5. RT/PR/IN genotype

Introduction

What this is:§For new clinicians (not experts)§Focus on

•Currently-used ARVs•Common resistance scenarios•Genotype tests•Approaches to interpreting R test results

§Proviral DNA genotype: a few words

Mechanisms of HIV Drug Resistance

§High rate of HIV replication- 109 virions per day§Many mutations and quasi species

• RT: error-prone, no copyediting§In setting of drug pressure with viral replication, selection

of resistant viruses• <- Inadequate adherence to ART• <- Wrong ARVs (potency), wrong doses (drug levels), drug-

drug interactions, absorption issues, etc.

• (Corollary: in absence of drug pressure, possible “disappearance” of mutations [minority populations])

Bottom Line: Assume that once there, always there (archived)

Mechanisms of HIV Drug Resistance

§ Mutations may decrease susceptibility to ARVs, and cause or contribute to virologic failure

• (a few mutations may increase susceptibility)§ Some mutations may impair viral fitness§ Some single mutations severely impact certain ARVs

(eg, M184I/V)§ In some cases, several mutations are needed to cause a

significant impact, esp. with NRTIs and PIs (eg, TAMs)§ Some ARVs may retain residual activity (eg, 3TC/FTC)§ Cross-resistance within an ARV class is common

When to do Resistance Test: DHHS Recommendations§ Before ART -- at first visit (as close to the time of

infection as possible)• Resistance mutations more likely to be detected earlier in

the course of HIV infection• GT: RT, PR; IN if concern for transmitted INI resistance

§ Virologic failure -- do on failing ARVs or shortly after d/c (w/in 4 weeks)

• GT for 1st or 2nd ART, add PT if “known or suspected complex drug resistance pattern”

• IN GT if virologic failure on INI

§ Suboptimal virologic response on ART

§ PregnancyDHHS Adult/Adolescent ART Guidelines 2018

Case: Transmitted Drug Resistance

•31 yo man with new diagnosis of HIV, chronicity unknown (no previous HIV test)

•CD4: 525, HIV RNA: 93,000 c/mL•GT: RT - M41L, K103N; PR – L63P

Prevalence of Transmitted HIV Drug Resistance in US, 2006-2009

Genotypic analysis of samples from newly diagnosed patients in CDC National HIV Surveillance System (N = 12,668)

Ocfemia MCB, et al. CROI 2012. Abstract 730.Menza TW et al, AIDS 2017.

All cases with sequencesCases classified as recent infectionsCases classified as long-standing infections

0

4

Transmitted Drug Resistance Mutations (TDRMs)

1 or more

20

8

12

16

1-class 2-class 3-class NNRTI NRTI PI

15.6%

7.86.8

4.1

INSTI: case reports, but no significant transmitted resistance—0.2% in one study in N. Carolina

Transmitted HIV Drug Resistance in MSM, 2010-2012

Banez Ocfemia, XXIV HIV Drug Resistance Workshop, 2015.

Newly diagnosed MSM patients age ≥13 in CDC National HIV Surveillance System, 8 jurisdictions (N = 9,629)

Genotype Test

§Standard GT examines RT and PR§For IN, must order special test§With GT, often possible to predict/anticipate resistance depending on the specific mutations, but is not a direct measure of resistance

Genotype Test

§C, cysteine§A, alanine§D, aspartate§E, glutamate§F, phenyalanine

§G, glycine§H, histidine§I, isoleucine§K, lysine§L, leucine

§M, methionine§N, asparagine§P, proline§Q, glutamine§R, arginine

§S, serine§T, threonine§V, valine§W, tryptophan§Y, tyrosine

M184V§First letter: WT amino acid§Number: codon position§Second letter: mutant amino acid

Phenotype Test

§ Measures inhibition of viral replication by individual ARVs in vitro

§ IC50 = concentration of drug required to inhibit viral replication by 50%

§ Compares patient virus IC50 to that of a reference strain, -> fold-change in IC50 relative to the reference strain

FC = IC50 patientIC50 reference

Phenotype (with GT)

Limitations of GT and PT

§ Conventional tests reliably detect mutant virus that comprises about 20% of the circulating viral quasispecies

§ May miss minority populations§ HIV RNA must be >500-1,000 c/mL§ Do not assess interaction of ARVs

NRTI Resistance

§ A number of key mutations for 3TC/FTC, TDF/TAF, ABC; + numerous others

§ Some single mutations severely impact viral replication, but with others an accumulation of mutations usually required for high-level resistance

• (More = worse)

§ NRTI Strategy: • Know key mutations (M184V, K65R, TAMs)• Look up others

NRTI Resistance:M184I/V§ Selected by 3TC, FTC (sometimes ABC), cause

resistance to 3TC, FTC§ Very common -- 3TC/FTC have low genetic barrier to

resistance§ Cross resistance: decreases susceptibility to ABC, ddI

(especially if TAMs)§ Increases susceptibility to TDF/TAF, AZT, d4T

• Partially restores activity if TAMs present

§ Decreases viral fitness

NRTI Resistance:K65R

§ Selected by TDF/TAF, ABC, ddI§ Causes resistance to TDF/TAF, all other NRTIs except

AZT§ Increases susceptibility to AZT§ Decreases viral fitness

NRTI Resistance:TAMs

Thymidine-associated mutations (TAMs)§ Selected by AZT, d4T§ Decrease susceptibility to AZT, d4T, TDF/TAF, ABC; to all

NRTIs if numerous§ Cross resistance: increases with increasing number of

TAMs• (More = worse)

§ 2 pathways: • M41L, L210W, T215Y (more resistance; incl. more

impact on TDF)• D67N, K70R, T215F, K219Q/E

NRTI Resistance:L74V/I

§ Selected by ABC and ddI• (L74V sometimes selected by TDF)

§ Resistance to ABC and ddI§ Increases susceptibility to TDF/TAF, AZT

Case 1: PrEP Failure

§ 31 yo MSM on PrEP (TDF/FTC, Truvada), presents to clinic after absence of 6 months, reports spotty adherence to his PrEP, but continues to take.

§ HIV Ab+, HIV RNA 65,000 c/mL.

§ HIV genotype: RT – K65R, M184V; PR – WT.

NRTI Strategy: • Know key mutations

(M184V, K65R, TAMS)• Look up others

Case 2: Audience Response

Which ART regimen would be most likely to be effective?

1. Rilpivirine (RPV)/TAF/FTC (Odefsey)2. EVG/cobi/TAF/FTC (Genvoya)3. DTG/ABC/3TC (Tivicay)4. DRV/r + rilpivirine/TAF/FTC

RT – K65R, M184VPR – WT

Bottom Line: need 3 active ARVs, if possible (especially if ARVs have low genetic barrier to resistance).

NNRTI Resistance

§ Several single mutations confer high-level resistance to certain NNRTIs; numerous others contribute to resistance

§ Low genetic barrier to resistance (except etravirine, ?doravirine)

§ Cross resistance is common

§ NNRTI Strategy: • Know several key mutations (K103N, Y181///, E138K

[V106])• Look up others• Be aware of mutation scoring system for etravirine (ETR) • (Be cautious about DOR – little is known)

NNRTI Resistance: Important Mutations§ K103N

• Commonly selected by EFV• Emerges early in VF (low genetic barrier to resistance), commonly

transmitted• Resistance to EFV, NVP• By itself, does not decrease susceptibility to ETR, RPV, DOR

§ Y181I/V/C/F/G/S• Selected by NNRTIs• Cross resistance to all NNRTIs (except DOR?)

§ E138K• Selected by RPV• Usually occurs with M184I or M184V; this enhances resistance to RPV• Resistance to RPV, cross resistance to EFV, ETR

§ V106I/ /• Frequently seen after DOR failure, with other mutations

Doravirine

• In vitro, active against common NNRTI resistance mutations (incl K103N, E138K, Y181C, G190A)

• In vivo, emergent resistance: • Treatment-naïve trials: V106I,

Y188L, H221Y, P225H, F227C • ALSO NRTI resistance

mutations – eg, M184V, K65R, M41L• Switch study (DRIVE-SHIFT):

none• NO clinical data in salvage

settings

LAI M-T. CROI 2016. Abs 506

Case 2: NNRTI Resistance

• 53 yo man with VF years ago on EFV/TDF/FTC (Atripla), now on dolutegravir/ABC/3TC (Triumeq); VL <40 c/mL x 1 year. He complains of headache and GI symptoms and wants to change ARVs.

• Previous GT (while on EFV/TDF/FTC):

Case 2: NNRTI Resistance§ Could we use rilpivirine (or doravirine) + 2 NRTIs

as his next ART regimen? • K103N alone should not affect RPV, DOR, or ETR

§ Issues:• Accuracy of GT?• Mutations may fade from view with time and

removal of selective drug pressure• GT captures strains that comprise >5-20% of the

circulating viruses• Once there, always there: “archived” mutations

White, KL. Toronto 2013. Abs 87.

Bottom Line: caution in interpreting GTs – consider what may be hidden from view

Case 3: 1st ART Failure

§ 35 yo woman on RPV/TAF/FTC (Odefsey). HIV RNA suppressed x 2 years, then increases to >5,000 c/mL in setting of several months of OTC PPI use.

§ Genotype: • RT - K101E, E138K, Y181C, M184I

• PR - no mutations

Case 3: Audience Response

Could you use etravirine (ETR) or doravirine (DOR) in her next regimen?

1. Yes – should have full activity2. No – not effective after rilpivirine failure3. I need more information

RT: K101E, E138K, Y181C, M184IPR: none

NNRTI Strategy: •Know several key mutations (K103N, Y181///, E138K [V106])•Look up others•Mutation scoring system for ETR•Caution re DOR – little is known)

Etravirine ResistanceMutation Weight Factor

Vingerhoets J, et al. AIDS. 2010;24:503-14. Tambuyzer et al JAIDS. 2011.

Genotypic score

%Responders(DUET trial)

0-2 74.4%2.5-3.5 52%≥4 38%

Response by ETR weight

Case: RT - K101E, E138K, Y181C, M184I

Bottom line: ETR may not be effective if ETR score >2.

1.0 V90I A98G K101E/H138G/K/Q 179D/TG190A

1.5 V106I E138A V179F G190S

2.5 L100I K101P Y181C M230L

3 Y181I Y181V

Integrase Inhibitor Resistance

§ Raltegravir and elvitegravir have low-ish barriers to resistance

• Several possible mutation pathways• Different effects on dolutegravir, ?bictegravir

§ Dolutegravir and bictegravir primary resistance is rare, not well understood

§ Order integrase genotype (not phenotype) = special order

• Important for predicting sensitivity to DTG, BIC

◆ Low et al, Antimicrob Agents Chemother. 2009;53:4275-82

Integrase Inhibitor Resistance

§ INSTI Strategy: • Assume cross-resistance between RAL and

EVG• Recognize Q148/// (most damaging to DTG)

• Look up all others

• (Be very cautious about BIC – little is known)

Low et al, Antimicrob Agents Chemother. 2009;53:4275-82

Integrase Inhibitor Resistance•

Low et al, Antimicrob Agents Chemother. 2009;53:4275-82.Kulkarni R et al, CROI 2013, Abs. 587.

Pathway/mutations Resistance

Raltegravir Q148H/K/R, G140S/A

N155H, E92Q

Y143R/H/C

All INIs (for DTG, Q148 + at least one other)

RAL, EVG

RAL

Elvitegravir E92Q

Q148H/K/R, G140S/A

T66I

N155H

EVG, RAL, low level DTG

All INIs (for DTG, Q148 + at least one other)

EVG

RAL, EVG

Integrase Inhibitor Resistance

Dolutegravir (DTG)§No significant reported emergent mutations if used in 3-drug regimen for initial therapy§Various mutations if used as monotherapy§Various mutations if used after RAL or EVG

§ Q148 H/K/R + others, N155H + others, R263K

Bictegravir (BIC)§No reported emergent mutations if used in 3-drug regimen for initial therapy§No data for use in salvage therapy

Case 4: INSTI Resistance § 50 yo man, on ATV/r/TDF/FTC for years, switched to

EVG/cobi/TAF/FTC (Genvoya) to simplify his ART§ VL remained <40 c/mL x 9 months, then increased to 7,200

c/mL§ GT (incl. integrase) done:

Case 4: INSTI Resistance

Will dolutegravir be potent in his salvage regimen?

1. Yes, these elvitegravir mutations do not affect DTG2. No, full cross resistance is likely3. Possibly, if we increase the dose of DTG to 50 mg BID

IN: G140S, Q148R

INSTI Strategy: •Assume cross-resistance between RAL and EVG•Recognize Q148/// (most damaging to DTG)•Look up all others•(Caution re BIC – little is known)

Dolutegravir ResistanceViking-3 Study: Virologic response lowest in patients with Q148 + ≥2 secondary mutations

Castagna et al, JID 2014:210: 354-362

VIKING-3: DTG 50mg BID + OBR in patients with INSTI failure + 2 class resistance; had at least one fully active drug in OBR

79%

59%

24%

*Included primary INI-resistance mutations N155H, Y143C/H/R, T66A or E92Q or only historical evidence of resistance^Secondary mutations from G140A/C/S, E138A/K/T or L74I.

*

^

Bottom Line: • If Q148 + 0 or

1 other mutation, DTG may be effective (with other active ARVs)

• Use BID dosing

Bictegravir: in vitro activity against resistant virus

Tsiang M, Antimicro Agents Chemother, 2016Bottom Line: BIC very little information on resistance

PI Resistance

§ Primary/Major mutations• Emerge first, decrease antiviral effect

§ Secondary/Minor mutations• Emerge later, increase fitness of strains with primary

mutations or further decrease antiviral effect§ Specific primary and secondary PI mutations,

depending upon PI§ **Accumulation of mutations usually required

for high-level resistance§ Cross resistance is complex

PI Resistance

§ PI Strategy:•Be aware of (but don’t memorize) list of DRV resistance-associated mutations (RAMs)

•Look up all others

DRV Resistance-Associated Mutations (DRV-RAMs)

§ 11 mutations associated with resistance to DRV:• V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V,

L89V

§ Once-daily dosing possible if no DRV-RAMS (ODIN study)

§ Twice-daily dosing recommended if ≥1 DRV-RAM

§ DRV response diminished with ≥3 DRV-RAMs

De Meyer et al, AIDS Res Hum Retroviruses. 2008;24:379-388Cahn et al, AIDS. 2011;25:929-39

Case 5: PI (and other) Resistance

§48 yo man, reports that he has been on “everything” in the past (except INSTIs), sometimes with poor adherence.§Historic genotype(s):

• PR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

Case 5: PI resistance

§ Assess PI resistance:• Check for DRV resistance-mutations

• One DRV-RAM: L33F -> DRV/r likely to be effective but should be given BID

• Look up all other mutations

Case: RT -- M41L, D67N, L210W, T215Y; V106I, Y181VPR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V

Case 5: Multiclass Resistance, use strategies

§48 yo man, reports that he has been on “everything” in the past (except INSTIs), sometimes with poor adherence.

§Historic genotype(s):• RT -- M41L, D67N, L210W, T215Y; V106I, Y181V• PR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

Approaching this: be methodical, apply strategies• NRTI • NNRTI • PI• (IN)

Case 5: Multiclass Resistance (2)

§Historic genotype(s):• RT -- M41L, D67N, L210W, T215Y; V106I, Y181V• PR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

Assess NRTI resistance:• 4 mutations (all TAMs) - affect TDF/TAF, ABC• Anything missing?

• No M184 V/I – why???NRTI Strategy: • Know key mutations

(M184V, K65R, TAMS)• Look up others

Case 5: Multiclass Resistance (3)

§ Historic genotype(s):• RT -- M41L, D67N, L210W, T215Y; V106I, Y181V• PR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

• Assess NNRTI resistance:• 2 NNRTI mutations (incl. Y181)• ETR score:

• 4.5• DOR: little known (caution: V106I)

1.0 V90I A98G K101E/H 138G/K/Q179D/TG190A

1.5 V106IE138A V179F G190S

2.5 L100I K101P Y181C M230L

3 Y181I Y181V

ETR Mutation Weight Factor

NNRTI Strategy: •Know several key mutations (K103N, Y181///, E138K [V106])•Look up others•Mutation scoring system for ETR•Caution re DOR – little is known)

Case 5: Multiclass Resistance (4)

§ Historic genotype(s):• RT -- M41L, D67N, L210W, T215Y; V106I, Y181V• PR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

• Assess PI resistance:• 8 PI mutations (1 DRV mutation)

PI Strategy:• Be aware of list of DRV

resistance-associated mutations (RAMs)

• Look up all others

Case 5: Multiclass Resistance (5):“Look up other mutations”

Stanford HIV Drug Resistance Database http://hivdb.stanford.edu

Case: RT -- M41L, D67N, 210W, T215Y; V106I, Y181VPR -- L10I, L33F, M36I/M, M46L, I54V, L63S, I64V, V82A

Conclusion: likely resistance to all NNRTIs (??DOR), and to all PIs except DRV – use BID

Case 5, continued

• Summary:• 3-class resistance:

• NRTI: 4 mutations (TAMs): M41L, D67N, 210W, T215Y• Cross resistance to TDF, ABC• No M184V/I but we will assume it is there

• 3TC/FTC resistance • NNRTI: 2 mutations: V106I, Y181V

• Likely resistance to ETR, DOR• PI: 8 mutations (1 DRV RAM)

• Integrase inhibitors: never exposed• What ARVs should we use in the next regimen?

What ARVs should we use in the next regimen?

§ Integrase inhibitor?§ Darunavir/r? § NNRTI?§ NRTIs? § CCR5 antagonist?

Proviral DNA Genotype

Background:§Standard resistance test requires plasma HIV RNA of >500-1,000 c/mL§Many patients currently on ART with VL <40 may have resistance from previous regimens but have no available resistance tests to guide ART changes (eg, simplification)§Can we do resistance testing on archived HIV?

Proviral DNA Genotype

§ Amplifies cell-associated HIV-1 proviral DNA from infected cells (PBMCs)

•Whole blood samples§ Analyzes HIV-1 polymerase region by new

generation sequencing technology

Case 6

• 55 yo man HIV+ 1993, on ART x years• Previous ART history unclear – remembers AZT, D4T, 3TC, ABC,

DDI, EFV, NVP, IDV, SQV, LVP-r

• Transferred to this clinic on ATV-r + AZT + TDF/FTC, HIV RNA = ND• Changed to ATV-r + RAL + TDF/FTC x 5 years, HIV RNA = ND

• Proviral DNA GT:• RT: M184V, K103K/N• IN: None• PR: L90L/M• Changed to EVG/cobi/TAF/FTC + DRV

Proviral DNA Genotyping – How Accurate?

Toma J. ICAAC. 2015

DNA Genotype

Bottom Line:

• Can be helpful if shows resistance mutations.

• May miss some or all existing resistance mutations: caution re negative results

• No clinical data

Summary

§ Do resistance tests at time of diagnosis and if virologic failure + suspected resistance (usually GT)

§ Resistance: once there always there (archived)§ Beware of what you don’t see (minority populations)§ Look at old resistance reports, treatment history§ Read resistance reports methodically § Caution re ARVs with scanty data (eg, BIC, DOR)§ **Seek expert advice**

§ Goal of treatment: suppressed HIV RNA for all

Resources

§ Stanford HIV Drug Resistance Database• http://hivdb.stanford.edu

§ IAS-USA HIV Drug Resistance Mutations Figures

and User Notes • https://www.iasusa.org

§ Clinician Consultation Center• (800) 933-3413

Monday – Friday, 9 a.m. – 8 p.m. EST

(Free!)